Monoclonal antibodies in which selected amino acids have been mutated to cysteine (i.e., engineered cysteine mAbs, or ecmAbs) are particularly suitable for use in conjugates (e.g., antibody drug conjugates (ADCs)) because the conjugates derived from them can have favorable properties including homogeneity, favorable pharmacokinetics, stability, and solubility. The cysteine mutations are placed in locations in the amino acid sequence of the antibody which generally do not form inter- or intra-chain disulfide bonds, and expression machinery inside the cell producing the mutant mAb treats the cysteine residues as unpaired cysteines. Consequently, the engineered cysteines are generally expressed in the form of mixed disulfides with non-encoded cysteine molecules (i.e., the engineered cysteines are “capped” with capping agents, e.g., cysteine, cysteinyl glycine, or glutathione).
Hence, in order to prepare conjugates (e.g., ADC) from ecmAbs, it is generally necessary to subject the ecmAb to reducing conditions to convert the engineered cysteine from a mixed disulfide to a free thiol, and typically this uncapping or “activation” is accompanied by reduction of the ecmAb inter-chain disulfides. Although inter-chain disulfides can be re-formed by mild oxidation, such re-oxidation steps add to the complexity and expense of ADC preparation. Selective reduction methods have been elusive, as it has proven difficult to reduce the engineered cysteines without simultaneously reducing the inter-chain disulfides. Consequently, selective reduction methods for uncapping of engineered cysteine residues are needed. The present invention addresses this and other needs.